Organoids, miniature tissues generated from self-organizing stem cells within three-dimensional (3D) extracellular matrices (ECM), have opened up exciting possibilities for in vitro studies of complex physiological processes. A key factor in the success of organoids is the use of a laminin-rich hydrogel derived from mouse tumors, Matrigel, to provide both physical support and biochemical ECM cues. However, Matrigel is a black box due to its complexity, composed of hundreds of components, and limited tunability. This makes it difficult to decipher the role of the ECM in controlling the various steps during organoid formation. One approach to explore the role of Matrigel and its key components is to recapitulate its properties using chemically defined and tunable ECM hydrogels. This thesis investigates the use of poly(ethylene)glycol (PEG)-based hydrogels that serve as well-defined Matrigel analogs. In Chapter1, I provide a brief overview and current challenges to set the research aims. As a model system to explore the role of the ECM in organoid-genesis, I focused on adult stem cell-derived intestinal organoids, a paradigmatic model system characterized by its multicellular complexity and patterning into crypt-villus domains. In Chapter2, I investigate the importance of the dynamic viscoelastic properties and in particular the stress relaxation of the native ECM during organoid formation. Here, I have developed hydrogels that are partially crosslinked via physical interactions through triple hydrogen bonds. In combination with covalent crosslinks, the hybrid hydrogel networks exhibited degradation-independent, stress-relaxing properties and were found to promote the first emergence of Paneth cells and thus the formation of crypt-villus structures. Laminin is essential for intestinal organoid formation, but its specific role in this process is unknown. In Chapter3, I therefore used synthetic matrices to systematically investigate the functional aspects of laminin-based ECM at different stages of intestinal organoid development, focusing on the effects of exogenous laminin. Using a blank slate matrix, the endogenous ECM produced by early epithelial cells in the developing organoid could be identified and spatially visualized. Moreover, I present a novel approach to isolate the specific ECM secreted by epithelial cells, thus creating for the first time a fully animal-free and organoid-specific culture system. In Chapter4, I explored the potential of synthetic matrices to control spatiotemporally biochemical cues that are equally important during organoid morphogenesis. As a proof of concept, I used photo-sensitive DNA modified with azobenzene to release signaling molecules at will within the hydrogel. The unique DNA sequences provided specificity that allowed simultaneous control over multiple signaling factors. With this versatile method, signaling gradients could be created in the 3D culture environment, allowing unprecedented control of organoid-genesis that normally occurs stochastically. Understanding the role of the complex ECM in organoid development has remained a major challenge in the field. In this work, synthetic ECM was introduced not only as an alternative to Matrigel but also as a powerful tool to study specific aspects of ECM biology and function. Overall, this work paves the way for the goal of developing more relevant and animal-free organoid culture systems that open up exciting prospects for translational applications.